a-Synuclein: A fusion chaperone significantly boosting the enzymaticperformance of PET hydrolase

Extensive use of polyethylene terephthalate (PET) has brought about global environmental problems. Arecently reported PET hydrolase (PETase) discovered from Ideonella sakaiensis showed high potentialfor degrading PET at moderate temperatures, but its activity and stability need further improvementfor practical applications. Herein, we proposed to use a-synuclein (aS) as a fusion chaperone and createdsix PETase-aS fusion enzymes with linkers of different types and lengths. All the fusion enzymes exhibited improved enzymatic performance, presenting 1.5 to 2.6-fold higher activity towards bis-2(hydroxyethyl) terephthalate than PETase, as well as significantly increased stabilities. Fluorescencespectroscopy indicated that the chaperone fusion tightened the overall conformation and resulted inthe opening of the substrate binding pocket, which led to the improved thermal stability and catalyticactivity of the fusion enzymes. Remarkably, one of the fusion proteins, PETase-[(GS)(EK)]10-aS, showed3.2 to 5.1 times higher PET degradation capability than PETase. The significantly boosted PET degradationperformance was not only attributed to the enhanced enzymatic activity and stability, but also possiblydue to the binding affinity of the fused aS domain for PET. These findings demonstrated that aS was aneffective fusion chaperone for significantly enhancing the enzymatic performance of PETase.

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alphasynuclein

The specific and effective a-synuclein RNA interference （RNAi） plasmids, and the a-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 （HEK293） cells using the lipofectamine method. Using an inverted fluorescence microscope, a-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type a-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies （Lewy body-like inclusions） in the cytoplasm of some cells transfected with a-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type a-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type a-synuclein in mitochondria.

Decreasing a-synuclein aggregation by methanolic extract of Centella asiatica in zebrafish Parkinson's model

Objective: To observe the effects of Centella asiatica(C. asiatica) methanolic extract on a-synuclein aggregation and its expression in rotenone-exposed zebra fish.Methods: Zebra fish(Danio rerio) were exposed to 5 m g/L rotenone for 28 days and coincubated with 2.5, 5.0 and 10.0 m g/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebra fish were sacrificed on the ice block and protein was isolated from zebra fish brain for ELISA of dopamine and Western blotting of a-synuclein. Immunohistochemistry was conducted to observe the a-synuclein expressions from histopathological preparation of zebra fish brain. The head were soaked in 10% formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using anti a-synuclein antibody. We also measured zebra fish motility for 5 min in each week.Results: C. asiatica has important bioactive compounds such as asiaticoside that has antiin flammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack to a-synuclein protein that caused aggregation and increase of its expression.The motility of zebra fish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebra fish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increased a-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebra fish, but C. asiatica increased dopamine level.Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson's such as a-synuclein aggregation and expression, increasing motility and dopamine as well.

Propofol Attenuates a-Synuclein Aggregation and Neuronal Damage in a Mouse Model of Ischemic Stroke

a-Synuclein is a soluble monomer abundant in the central nervous system. Aggregates of a-synuclein,consisting of higher-level oligomers and insoluble fibrils,have been observed in many chronic neurological diseases and are implicated in neurotoxicity and neurodegeneration.a-Synuclein has recently been shown to aggregate following acute ischemic stroke, exacerbating neuronal damage.Propofol is an intravenous anesthetic that is commonly used during intravascular embolectomy following acute ischemic stroke. While propofol has demonstrated neuroprotective properties following brain injury, the mechanism of protection in the setting of ischemic stroke is unclear. In this study, propofol administration significantly reduced the neurotoxic aggregation of a-synuclein, decreased the infarct area, and attenuated the neurological deficits after ischemic stroke in a mouse model. We then demonstrated that the propofol-induced reduction of a-synuclein aggregation was associated with increased mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 signaling pathway activity and reduction of the excessive autophagy occurring after acute ischemic stroke.

The Concentration of Hydrogen Peroxide Generated during Aggregation of a-Synuclein in vitro Is Lower than 5 nmol/L

Using a fluorometric method with a detection limit of 5 nmol/L, here it is reported that albeit positive results were got from bovine serum albumin (BSA) and chicken ovalbumin (OVA) as published in literature, no detectable amount of hydrogen peroxide (H2O2) was generated during a-synuclein (a-Syn) aggregation in vitro even in the presence of transition metal ions Cu(II) or Fe(III). The results suggest that the concentration of H2O2 generated during aggregation of a-Syn in vitro be lower than 5 nmol/L beyond the detection limit of the adopted method and it is far too poor to be responsible for the cytotoxicity of a-Syn aggregates, thus allowing people to extensively elu-cidate the mechanism underlying neurotoxicities of the aggregates formed by some amyloidogenic proteins.

突触核蛋白病中α-syn聚集及播散的机制研究

突触核蛋白病（α-synucleinopathy）是一类以大脑中不同类型α-突触核蛋白（α-synuclein,简称α-syn）阳性包涵体为病理特征的神经变性疾病,包括帕金森病、路易体痴呆、多系统萎缩及其他含α-突触核蛋白病理的少见疾病。其中,帕金森病和路易体痴呆的标志是脑干或皮质Lewy小体,而多系统萎缩则表现为少突胶质细胞和神经元的胞浆包涵体[1]。

自噬在帕金森病中的研究进展

帕金森病（Parkinson＇s disease,PD）是神经系统第二大变性病,影响着世界约5千万人口。Karin Wirdefeldt等在总结了世界各地流行病学资料后于2011年发表了1篇文献中指出PD的发病率为（1.5～22）/10万人年,其患病率约为（167～5 703）/10万。

Neuroprotective effects of genistein on SH-SY5Y cells overexpressing A53T mutant α-synuclein

Genistein, a potent antioxidant compound, protects dopaminergic neurons in a mouse model of Parkinson＇s disease. However, the mecha- nism underlying this action remains unknown. This study investigated human SH-SYSY cells overexpressing the A53T mutant of α-synuclein. Four groups of cells were assayed： a control group （without any treatment）, a genistein group （incubated with 20 μM genistein）, a rote- none group （treated with 50 μM rotenone）, and a rotenone ＋ genistein group （incubated with 20 μM genistein and then treated with 50μM rotenone）. A lactate dehydrogenase release test confirmed the protective effect of genistein, and genistein remarkably reversed mitochondrial oxidative injury caused by rotenone. Western blot assays showed that BCL-2 and Beclin ! levels were markedly higher in the genistein group than in the rotenone group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed that genistein inhibited rotenone-induced apoptosis in SH-SYSY cells. Compared with the control group, the expression of NFE2L2 and HMOX1 was significantly increased in the genistein ＋ rotenone group. However, after treatment with estrogen receptor and NFE2L2 channel blockers （ICI-182780 and ML385, respectively）, genistein could not elevate NFE2L2 and HMOX1 expression. ICI-182780 effectively prevented genistein-mediated phosphorylation of NFE2L2 and remarkably suppressed phosphorylation of AKT, a protein downstream of the estrogen receptor. These findings confirm that genistein has neuroprotective effects in a cell model of Parkinson＇s dis- ease. Genistein can reduce oxidative stress damage and cell apoptosis by activating estrogen receptors and NFE2L2 channels.

Natural polyphenols effects on protein aggregates in Alzheimer＇s and Parkinson＇s prion-like diseases

Alzheimer＇s and Parkinson＇s diseases are the most common neurodegenerative diseases. They are characterized by protein aggregates and so can be considered as prion-like disease. The major components of these deposits are amyloid peptide and tau for Alzheimer＇s disease, α-synuclein and synphilin-1 for Parkinson＇s disease. Drugs currently proposed to treat these pathologies do not prevent neurodegenerative processes and are mainly symptomatic therapies. Molecules inducing inhibition of aggregation or disaggregation of these proteins could have beneficial effects, especially if they have other beneficial effects for these diseases. Thus, several natural polyphenols, which have antioxidative, anti-inflammatory and neuroprotective properties, have been largely studied, for their effects on protein aggregates found in these diseases, notably in vitro. In this article, we propose to review the significant papers concerning the role of polyphenols on aggregation and disaggregation of amyloid peptide, tau, α-synuclein, synphilin-1, suggesting that these compounds could be useful in the treatments in Alzheimer＇s and Parkinson＇s diseases.

Ginsenoside Rg1 attenuates motor impairment and neuroinflammation in the MPTP-probenecid-induced parkinsonism mouse model

OBJECTIVE To evaluate these activities of Rg1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)/probenecid(MPTP/p)-induced PD mouse model for the first time and to elucidate the underlying mechanisms.METHODS Male C57BL/6 mice were randomly assigned to six groups.One hour prior to MPTP/p injection,GroupⅢ-Ⅵmice received 10 mg·kg^(-1),20 mg·kg^(-1),or 40 mg·kg^(-1) Rg1 or 3 mg·kg^(-1) selegiline,respectively,orally from D(-3) to D49.GroupⅠ-Ⅱmice received solvent water.Subsequently,GroupⅡ-Ⅵmice received by injection MPTP-HCl(25 mg·kg^(-1) bw dissolved in0.9%saline,sc)on a 40-d schedule at intervals of 4 d between consecutive doses in combination with an adjuvant drug,probenecid(250 mg·kg^(-1) bw in 0.03 mL of DMSO,ip);GroupⅠmice were injected with saline and probenecid.Behavioral performance was assessed in the open field test,pole test and rotarod test.Neurotransmitters in the striatum were detected using HPLC.Protein levels were measured by Western blot.Pathological characteristics were examined by immunohistochemistry.Ultrastructure changes were observed by electron microscopy.RESULTS Oral treatment with Rg1 significantly attenuated the high MPTP-induced mortality,behavior defects,loss of dopamine neurons and abnormal ultrastructure changes in the SNpc.Other assays indicated that the protective effect of Rg1 may be mediated by its anti-neuroinflammatory properties.Rg1 regulated MPTP-induced reactive astrocytes and microglia and decreased the release of cytokines such as tumor necrosis factor-α(TNF-α)and interleukin-1b(IL^(-1)b)in the SNpc.Rg1 also al eviated the unusual MPTP induced increase in oligomeric,phosphorylated and disease-related a-synuclein in the SNpc.CONCLUSION Rg1 protects dopaminergic neurons,most likely by reducing aberrant a-synuclein-mediated neuroinflammation,and holds promise for Parkinson disease therapeutics.

synphilin-1在帕金森病发病中的作用

α-synuclein蛋白是帕金森病(PD)lewy小体(LB)的主要成分,近年在LB中又发现一新的蛋白synphilin-1,且与α-synulein相互作用。有研究认为synphilin-1可能在LB的形成中起作用。了解synphilin-1的结构、功能与PD及相关蛋白的关系,可进一步探讨PD的发病机理。

帕金森病相关基因的研究进展

近年来随着分子生物学技术的应用和对帕金森病病因学的深入研究 ,遗传因素在疾病发生、发展过程中的作用已成为许多学者关注的焦点。同一环境因素下 ,相关基因的多态性与帕金森病易患性的关系也日益受到重视。本文就可能与帕金森病有关的基因作一综述。

Structural changes of gut microbiota in Parkinson's disease and its correlation with clinical features

The aim of this study was to compare the structure of gut microbiota in Parkinson's disease(PD) patients and healthy controls;and to explore correlations between gut microbiota and PD clinical features. We analyzed fecal bacterial composition of 24 PD patients and 14 healthy volunteers by using 16 S rRNA sequencing. There were significant differences between PD and healthy controls, as well as among different PD stages. The putative cellulose degrading bacteria from the genera Blautia(P=0.018),Faecalibacterium(P=0.048) and Ruminococcus(P=0.019) were significantly decreased in PD compared to healthy controls.The putative pathobionts from the genera Escherichia-Shigella(P=0.038), Streptococcus(P=0.01), Proteus(P=0.022), and Enterococcus(P=0.006) were significantly increased in PD subjects. Correlation analysis indicated that disease severity and PD duration negatively correlated with the putative cellulose degraders, and positively correlated with the putative pathobionts. The results suggest that structural changes of gut microbiota in PD are characterized by the decreases of putative cellulose degraders and the increases of putative pathobionts, which may potentially reduce the production of short chain fatty acids, and produce more endotoxins and neurotoxins; and these changes is potentially associated with the development of PD pathology.

Insights into the pathogenesis of multiple system atrophy: focus on glial cytoplasmic inclusions

Multiple system atrophy(MSA)is a debilitating and fatal neurodegenerative disorder.The disease severity warrants urgent development of disease-modifying therapy,but the disease pathogenesis is still enigmatic.Neurodegeneration in MSA brains is preceded by the emergence of glial cytoplasmic inclusions(GCIs),which are insoluble α-synuclein accumulations within oligodendrocytes(OLGs).Thus,preventive strategies against GCI formation may suppress disease progression.However,although numerous studies have tried to elucidate the molecular pathogenesis of GCI formation,difficulty remains in understanding the pathological interaction between the two pivotal aspects of GCIs;asynuclein and OLGs.The difficulty originates from several enigmas:1)what triggers the initial generation and possible propagation of pathogenic α-synuclein species?2)what contributes to OLG-specific accumulation of α-synuclein,which is abundantly expressed in neurons but not in OLGs?and 3)how are OLGs and other glial cells affected and contribute to neurodegeneration?The primary pathogenesis of GCIs may involve myelin dysfunaion and dyshomeostasis of the oligodendroglial cellular environment such as autophagy and iron metabolism.We have previously reported that oligodendrocyte precursor cells are more prone to develop intracellular inclusions in the presence of extracellular fibrillary α-synuclein.This finding implies a possibility that the propagation of GCI pathology in MSA brains is mediated through the internalization of pathological α-synuclein into oligodendrocyte precursor cells.In this review,in order to discuss the pathogenesis of GCIs,we will focus on the composition of neuronal and oligodendroglial inclusions in synucleinopathies.Furthermore,we will introduce some hypotheses on how α-synuclein pathology spreads among OLGs in MSA brains,in the light of our data from the experiments with primary oligodendrocyte lineage cell culture.While various reports have focused on the mysterious source of α-synuclein in GCIs,insights into the mechanism which regulates the uptake of pathological α-synuclein into oligodendroglial cells may yield the development of the disease-modifying therapy for MSA.The interaction between glial cells and asynuclein is also highlighted with previous studies of post-mortem human brains,cultured cells,and animal models,which provide comprehensive insight into GCIs and the MSA pathomechanisms.